We have previously shown a time-of-day variation in the response of systolic [Ca²⁺]_i to the non-specific ß-adrenergic (ß-ADR) agonist isoproterenol (ISO), which is linked to a variation in Nitric Oxide (NO) signalling [1]. This may reflect stimulation of ß₃-ADRs, which induces a NO-dependent negative inotropic response [2]. As action potential duration (APD) impacts systolic [Ca²⁺]_i and a time-of-day variation exists in the regulation AP [3], we investigated the effect of ß₃-ADR stimulation on the APD and systolic [Ca²⁺]_i.

Ventricular myocytes were isolated by enzymatic digestion of male Wistar rat hearts at two time points; 3 h into the rest-period and active-period. [Ca²⁺]_i was measured in myocytes loaded with Fura-2. APD was measured using whole-cell patch-clamp recording. I _to and I _LTCC current density were measured using the whole-cell voltage-clamp technique. Data shown as number of hearts (number of cells), mean ± S.E.M., 2-way ANOVA, Tukey’s post hoc test.

In order to determine whether the previously shown time-of-day variation in response to ISO can be explained by a variation in ß₃-ADR dependant NOS signalling, we investigated systolic [Ca²⁺]_i in rest and active-period myocytes in response to the specific ß₃-ADR agonist BRL37344. BRL37344 (200 nM) resulted in a significant reduction in systolic [Ca²⁺]_i in rest-period myocytes, from 458.5 ± 41.2 nM to 361.2 ± 18.0 nM (n = 3–4 (18–19), P < 0.001) but had no significant effect on active period myocytes.

BRL37344 also significantly reduced APD₃₀, 18.3 ± 2.2 ms to 14.4 ± 1.6 ms (P < 0.001), and APD₅₀, 32.9 ± 4.3 ms to 26.5 ± 3.1 ms, (P < 0.001) in rest-period myocytes (n = 3–4 (12–15) with no significant change in either APD_30/50 in active-period myocytes.

I _to current density was measured in rest-period myocytes using K⁺ electrode solution; cell perfusate contained 200 nM CdCl^₂ to block Ca²⁺ currents. BRL37344 caused a significant drop in I _to density at +30 mV, from 6.38 ± 0.90 to 4.59 ± 0.11 pA/pF (n = 4 (13), P < 0.05). There was a small but non-significant recovery of the reduction due to BRL following NOS inhibition with L-NNA.

I _LTCC current density was measured in rest-period myocytes using Cs electrode solution to block K⁺ currents. Stimulation of ß₃-ADR with BRL37344 caused no significant difference in either peak LTCC current density, from -7.46 ± -0.50 to -8.09 ± -0.80 pA/pF, or in the voltage dependence of the peak Ca²⁺ current, 0mV (n = 3 (13)).

The time-of-day variation we have described previously is seen a decreased [Ca²⁺]_i sensitivity during the active-period. Therefore, our data suggests that ß₃-ADR dependant NOS signalling is not likely to be responsible for this time-of-day variation as BRL37344 caused a significant drop in [Ca²⁺]_i and APD_30/50 in rest-period myocytes only. Further investigation of the effect of BRL37344 on rest-period myocytes appears to show the reduction in [Ca²⁺]_i and APD_30/50 is not due to an increase in the I _to or to a reduction in I _LTCC.